10 Tips for Successful qPCR

Improving results performance by deeper understanding
of every qPCR step

High Quality RNA – RNA should either be prepared from fresh tissue, or from tissue treated with an RNA stabilization solution such as Invitrogen™ RNAlater® Stabilization Solution. See guideline for working with RNA

Good Primers and Probe Design – For optimal primers and probe design we strongly recommend using a dedicated software. Such software will take in consideration some adjustable parameters: primers/probe Tm, complementarity, and secondary structure as well as amplicon size and number of identical nucleotides runs. When designing amplicons in eukaryotic targets, choose PCR primers that span at least one exon-exon junction in the target mRNA to prevent amplification of the target from contaminating genomic DNA. If you want to make it much easier, choose a predesigned Applied Biosystems™ TaqMan® Assay. Each assay contains target primers and an optimized sequence-specific probe.

A Master Mix – using a master mix with all qPCR components mixed together, helps to minimize sample-to-sample and well-to-well variation and improves reproducibility. To further reduce well-to-well variation, use a master mix that contains a reference dye such as ROX. Use this helpful selection guide or contact our specialists at Rhenium qPCR Support to choose the right master mix for your experiment.

Use “No RT” Control – In addition to NTC, it is important to include a minus-reverse transcriptase control (“No Amplification Control” or NAC) in qPCR experiments. The NAC is a mock reverse transcription containing all the reagents, except the reverse transcriptase. If a product is seen in the NAC, it probably indicates that contaminating gDNA is present in the sample. It is mostly important to run a No-RT when primer design does not span an exon-exon junction site, so that genomic DNA may be detected.

Use an Appropriate Normalization Control – The reliability of any qPCR experiment can be improved by including an invariant endogenous control to correct for sample-to-sample variations. The expression level of a good control should be constant across the samples being analyzed. To learn more, you can read this newsletter on Endogenous Controls in qPCR.

Performing Melting Curve When Using SYBR® Green – Ideally, the experimental samples should yield a sharp peak at the melting temperature of the amplicon, whereas the NAC and NTC will not generate significant fluorescent signal. This result indicates that the amplified products are specific. If the melting curve reveals a series of peaks, it indicates that there is not enough discrimination between specific and non-specific reaction products. To obtain meaningful data, optimization would be necessary.

Setting the Baseline and Threshold Properly – To obtain accurate Ct values, the baseline needs to be set two cycles earlier than the Ct value for the most abundant sample. For real-time PCR data to be meaningful, the threshold should be set when the product is in exponential phase. To learn more, you can read this newsletter on THRESHOLD.

Confirming the Efficiency of the Reaction – The efficiency of the PCR should be 90 – 110%. Several variables can affect the efficiency, including length of the amplicon, secondary structure, and primers design. Read more about Efficiency.

Using an Appropriate Range for Standard Curves – Standard curves should be prepared for each gene under study for RNA quantitation (absolute or relative quantitation), or for verification of the efficiencies of the reactions for comparative quantitation (DDCt). The standard curve should extend above and below the expected abundance of your target. Additional input quantities can be included such as the minimum and maximum RNA amounts above and below the limit of detection to help differentiate between specific and non-specific products.